Kinetic Competition During the Transcription Cycle Results in Stochastic RNA Processing

Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate

Water vapour in the atmosphere of a transiting extrasolar planet

Water is predicted to be among, if not the most abundant molecular species after hydrogen in the atmospheres of close-in extrasolar giant planets (hot-Jupiters) Several attempts have been made to detect water on an exoplanet, but have failed to find compelling evidence for it or led to claims that should be taken with caution. Here we report an analysis of recent observations of the hot-Jupiter HD189733b taken during the transit, where the planet passed in front of its parent star. We find that absorption by water vapour is the most likely cause of the wavelength-dependent variations in the effective radius of the planet at the infrared wavelengths 3.6, 5.8 and 8 microns. The larger effective radius observed at visible wavelengths may be due to either star variability or the presence of clouds/hazes. We explain the most recent thermal infrared observations of the planet during secondary transit behind the star, reporting a non-detection of water on HD189733b, as being a consequence of the nearly isothermal vertical profile of the planet.s atmosphere. Our results show that water is detectable on extrasolar planets using the primary transit technique and that the infrared should be a better wavelength region than the visible, for such searches

author response kinetic competition during the transcription cycle results in stochastic rna processing

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